CN113874029B - Anticancer agents based on CP2C targeting peptides - Google Patents
Anticancer agents based on CP2C targeting peptides Download PDFInfo
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Abstract
The present invention relates to anticancer agents based on CP2c targeting peptides. The CP2 c-targeting peptide according to the present invention binds to the transcription factor CP2c in order to inhibit formation of a CP2 c-containing transcription factor complex (CP 2c homotetramer and CP2c/CP2b/PIAS1 heterohexamer), thereby inducing cancer cell-specific cell death, and a fatty acid is bound to the peptide to ensure stability enabling long-term maintenance thereof in vivo.
Description
Technical Field
The present invention relates to anticancer agents based on CP2c targeting peptides.
Background
Although many protein/peptide-based anticancer agents and low molecular compound-based anticancer agents have been developed and used, they do not selectively act only on cancer cells in vivo, but also exert serious side effects on normal cells, and their effects are sometimes insignificant due to the occurrence of drug-resistant cancer cells. In addition, many protein/peptide anticancer agents under development have a short half-life in vivo, and thus various studies are being conducted to overcome this problem. As such, despite worldwide competitive efforts to develop anticancer drugs, anticancer drugs having no problems in vivo stability, safety and drug resistance have not been developed yet. Thus, it has been found that a drug which ensures long-term stability in vivo, selectively removes only various types of cancer cells, and acts on drug-resistant cancer cells would be highly beneficial worldwide.
Disclosure of Invention
Technical problem to be solved
The present invention has been made in view of the above problems, and an important point thereof is to provide an anticancer agent based on CP2 c-targeting peptide, which is capable of ensuring long-term stability in vivo, selectively removing only various types of cancer cells, and also acting on drug-resistant cancer cells.
Technical proposal
According to the present invention, CP2 c-targeting peptide refers to a peptide that binds to a transcription factor CP2c to inhibit formation of a CP2 c-containing transcription factor complex (CP 2c homotetramer and CP2c/CP2b/PIAS1 heterohexamer), thereby inducing cancer cell-specific cell death. The CP2c targeting peptide corresponds to Tyr-Pro-Gln-Arg (SEQ ID NO: 1), which is the smallest size peptide consisting of only amino acids necessary for anticancer effect. Thus, peptides which essentially comprise these four amino acids and which exhibit an anticancer effect by interacting with CP2c proteins can be used as CP2 c-targeting peptides according to the present invention.
The CP2c targeting peptide according to the present invention may be a peptide consisting of 4 to 20 amino acids comprising the amino acid sequence of SEQ ID No. 1. Preferably, the CP2c targeting peptide according to the present invention may be a peptide (ACP 52) consisting of 6 amino acids (6 aa) 'NYPQRP (Asn-Try-Pro-Gln-Arg-Pro, SEQ ID NO: 2)'.
The CP2c targeting peptide according to the present invention may be conjugated to a cell penetrating peptide (CPP: cell-PENETRATING PEPTIDE) in order to enhance cell penetrating activity.
For example, a CP2c targeting peptide according to the present invention may be a peptide comprising a peptide consisting of SEQ ID NO:2 (ACP 52), and an internalizing RGD (internalizing RGD, iRGD) peptide consisting of 9 amino acids (9 aa) 'CRGDKGPDC (Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys, SEQ ID NO: 3)', as a Cell Penetrating Peptide (CPP). As one example of CP2C targeting peptide according to the present invention, 'ACP52C' is a peptide in which acetyl (Ac) is bound to the N-terminus of ACP52, igbd is bound to the C-terminus Pro, and an amide group (NH 2) is bound to the C-terminus of igdd (see fig. 7).
In addition, the present invention prepares CP2 c-targeting peptide-fatty acid conjugates that bind fatty acids for the purpose of ensuring in vivo stability of previously developed CP2 c-targeting peptides. When the fatty acid binds to the CP2c targeting peptide, the resulting conjugate binds to albumin in the blood, thereby improving in vivo stability and providing high therapeutic effect.
The fatty acid may be, but is not necessarily limited to, a C 12 to C 20 fatty acid. In one embodiment of the invention, the fatty acid may be a C 16 fatty acid, such as palmitoyl acid (also denoted as 'pal'). In addition, in one embodiment of the present invention, the fatty acid may be a modified fatty acid that binds to an amino acid sequence shown as glutamic acid (Glu, E) or EGLFG as a target sequence for proteolytic enzyme cathepsin B. In particular, the modified fatty acid may be formed by a peptide bond between the carboxyl group of the fatty acid and the amino group of glutamic acid or the amino group of glycine in the amino acid sequence shown in EGLFG. In the present specification, palmitoyl acid coupled to glutamic acid may be referred to as "E-pal", and palmitoyl acid coupled to the amino acid sequence shown in EGLFG may be referred to as "EGLFG-pal".
In addition, CP2 c-targeting peptide-fatty acid conjugates according to the present invention may comprise a CP2 c-targeting peptide (e.g., ACP 52) for CP2c targeting and a linker peptide for linking the CPP (e.g., igbd) and/or fatty acid to each other. The linker peptide may be an amino acid known in the art or a peptide composed of a combination thereof, without particular limitation. In particular, the linker peptide may comprise glycine (Gly, G), such as G n (where n is an integer from 1 to 6). In another embodiment of the invention, the linker peptide may consist of an amino acid sequence shown as G nKGm (where n and m are each independently integers from 0 to 6). For example, when N or m is 0, lysine (Lys, K) may be located at the N-or C-terminus of the linker peptide, and when N and m are not O, lysine (Lys, K) may be located between glycine. For conjugation between fatty acids and peptides, lysine (Lys, K) is included in the linker peptide. The terminal functional group of lysine (-HN 2) may enable the binding of additional amino acids.
In the CP2 c-targeting peptide-fatty acid conjugate according to the present invention, conjugation of the CP2 c-targeting peptide and the fatty acid may be achieved by a linker peptide. In particular, conjugation can be achieved by binding between glutamic acid as a peptide bonded to the modified fatty acid and lysine of the linker peptide. More particularly, the binding between glutamic acid of the modified fatty acid and lysine of the linker peptide may be formed by a peptide bond between the functional group (NH 2) of lysine and the carboxyl group of glutamic acid.
Meanwhile, the N-terminal and/or C-terminal of the peptide of the CP 2C-targeting peptide-fatty acid conjugate used in the present invention may be modified in order to obtain improved stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., broad spectrum of biological activity), and reduced antigenicity of the peptide used. Preferably, the modification may be in a form in which acetyl, fluorenylmethoxycarbonyl, amide, formyl, myristyl, stearoyl or polyethylene glycol (PEG) is bonded to the N-and/or C-terminus of the peptide, but may include, but is not limited to, modifications capable of improving the peptide, in particular the stability of the peptide. As used herein, the term "stability" means not only in vivo stability to protect the peptides of the invention from in vivo proteolytic enzyme attack, but also storage stability (e.g., room temperature storage stability). In the CP 2C-targeting peptide-fatty acid conjugate according to the present invention, the N-terminal and C-terminal of the peptide may be modified with acetyl and amide groups, respectively.
CP2 c-targeting peptide-fatty acid conjugates according to embodiments of the present invention are as follows:
1) Ac-K (E-pal) -GG-is bonded to the ACP52C N end where Ac is not present (C16-ACP 52Cn, ACP52 CG),
2) Ac-K (EGLFG-pal) -GG-is end-bonded to ACP52C N in which Ac is not present (A16-GFLG-ACP 52Cn, ACP52 CK),
3) The functional group of K in the GG-K-GG linker peptide located between ACP52 and iRGD is bound to the glutamic acid alpha-carbon carboxyl group of E-Pal (C-16-ACP 52Cm, ACP52 GK)
4) The functional group of K in the GG-K-GG linker peptide located between ACP52 and iRGD is bonded to the glutamic acid gamma-carbocarboxyl group of E-Pal (γC16-ACP52Cm, ACP52 CGK).
Deleting pictures
For example, the structures of ACP52GK and ACP52CGK in CP2 c-targeting peptide-fatty acid conjugates according to the invention are as follows:
In one example of the present invention, in vivo stability, anticancer effect, tumor metastasis inhibition effect and physiological toxicity in mouse models xenografted with hepatoma cells (Hep 3B) or breast cancer cells (MDA-MB-231) were analyzed using 4 synthetic CP2 c-targeting peptide-fatty acid conjugates. As a result, it was confirmed that the in vivo stability, tumor suppression effect and tumor metastasis suppression effect were excellent, and that the conjugate was a safe substance that was not toxic to the body.
In addition, it was confirmed that cells resistant to CP2 c-targeting peptide-fatty acid conjugates were generated during treatment with 4 synthetic CP2 c-targeting peptide-fatty acid conjugates to study anticancer effects in one example of the present invention. Thus, the mechanism and reason for resistance generation was demonstrated to be due to a decrease in cellular MDM2p90 levels in cells treated with CP2 c-targeted peptide-fatty acid conjugates. In addition, it was demonstrated that cancer cells resistant to CP2 c-targeting peptide-fatty acid conjugates can be killed by co-treatment of CP2 c-targeting peptide-fatty acid conjugates with caspase2 (caspase 2) inhibitors capable of inhibiting MDM2p90 degradation.
The present invention provides a pharmaceutical composition for preventing, ameliorating and treating cancer, which comprises a CP2 c-targeting peptide-fatty acid conjugate as an active ingredient.
As another aspect of the invention, the invention also provides a method of treating an individual, the method comprising the step of administering to an individual in need of treatment a CP2 c-targeting peptide-fatty acid conjugate, or a pharmaceutical composition comprising the same, in a pharmaceutically effective amount.
In the present invention, the cancer may further comprise a resistant cancer showing resistance to an anticancer agent, in particular an anticancer agent based on the CP2 c-targeting peptide-fatty acid conjugate according to the present invention.
In the present invention, the term "treatment" refers to any behavior that ameliorates or beneficially alters the symptoms of cancer by administering a peptide according to the present invention or a pharmaceutical composition comprising the peptide.
In the present invention, the term "administering" means introducing a predetermined substance, i.e. a derivative of a peptide according to the present invention or a pharmaceutical composition comprising the same, into a subject by any suitable method. The route of administration may be any general route as long as the drug can reach the target tissue. For example, the route of administration may include, but is not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration. However, since peptides are digested upon oral administration, it is preferred to formulate oral compositions coated with active agents or protected from degradation in the stomach. Preferably, the peptide will be administered in the form of an injection. In addition, the pharmaceutical composition according to the present invention may be administered by any means capable of transporting the active substance to the target cells.
In the present invention, the term "contained as an active ingredient" means an amount sufficient to treat a disease in a reasonable risk-benefit ratio applicable to pharmaceutical treatment. The effective dosage level may be determined based on the type of disease, severity, pharmaceutical activity, drug sensitivity, time of administration, route of administration, rate of excretion, duration of treatment, factors containing the concomitant drug and other factors well known in the medical arts. The peptide according to the present invention or the pharmaceutical composition comprising the same may be administered as a sole therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered one or more times. With all of the above factors in mind, it is important to administer the minimum amount that can achieve maximum effect and no side effects, which can be readily determined by those with medical expertise. The dosage and frequency of the pharmaceutical composition of the present invention are determined based on the type of drug as an active ingredient, together with several related factors such as the type of disease, the administration route, the age, sex and weight of the patient, and the severity of the disease.
The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease with a reasonable risk-benefit ratio applicable to pharmaceutical treatment. The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, age, weight and sex of patient, severity of disease, food, administration time and route, administration route, discharge rate and response sensitivity, and the ordinarily skilled physician can easily determine and prescribe an effective dosage for the desired treatment.
The term "individual" as used herein in the present invention includes animals, such as horses, sheep, pigs, goats, camels, antelopes, dogs or humans, the symptoms of which can be ameliorated by the administration of a therapeutic composition according to the present invention. Diseases can be effectively prevented and treated by administering the pharmaceutical composition according to the present invention to an individual. The treatment method according to the present invention may be a method of treating animals other than humans, but is not limited thereto. In other words, the composition according to the present invention may be sufficiently used for the treatment of a human in view of the fact that the human may have a disease whose symptoms may be ameliorated by the administration of the composition according to the present invention.
Thus, the pharmaceutical composition according to the present invention may comprise a variety of pharmaceutically acceptable carriers as long as the peptide according to the present invention is contained as an active ingredient. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants and flavoring agents for oral administration. The injection may be used by mixing a buffer, a preservative, an analgesic, a solubilizer, an isotonic agent or a stabilizer. For topical application, a matrix, excipient, lubricant or preservative may be used. The formulation of the pharmaceutical composition of the present invention may be prepared in a variety of ways by mixing with a pharmaceutically acceptable carrier as described above. For example, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups or wafers for oral administration, and it may be prepared in the form of a single dose ampoule or multiple doses for injection. It can also be prepared in the form of other solutions, suspensions, tablets, pills, capsules or sustained release formulations.
Meanwhile, some examples of carriers, excipients and diluents suitable for the formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil. In addition, fillers, anti-agglomerating agents, lubricants, wetting agents, flavoring agents or preservatives may also be used.
Hereinafter, the present invention will be described in more detail with reference to the following examples. It should be understood that the examples are included merely for purposes of illustration and are not intended to limit the spirit of the invention in any way.
ADVANTAGEOUS EFFECTS OF INVENTION
To identify the final candidate for increased in vivo stability of the transcription factor CP2C targeting peptide ACP52C (which shows an effect as a general anticancer agent), C 16 fatty acids were linked to CP2C targeting peptide precursor ACP52C and it was confirmed that the conjugate showed in vivo stability and anticancer effect in cancer cell xenograft mouse models. In addition, the cancer cell-specific anticancer effect of the final anticancer candidate was confirmed in various cancer cells and normal cells. The anticancer effect of the final candidate was shown to be caspase 2 dependent and its therapeutic effect on cancer cells resistant to anticancer drugs was confirmed by treatment with a combination of caspase 2 inhibitor and final candidate.
Drawings
Fig. 1 shows GI 50 values dependent on ACP52C treatment in various cancer cell lines.
Figures 2a to d show that ACP52C treatment induced G2/M cell cycle arrest and cell death (sub G1 cells), as shown by FACS.
Figures 3a through C show that ACP52C treatment results in an increase in p53 expression that regulates cell cycle and apoptosis.
Figures 4a through C show apoptosis induced by ACP52C treatment.
FIG. 5 shows time-dependent subcellular movement, localization and amount of ACP52C in MDA-MB-231 cell lines.
Fig. 6a to h show the in vivo half-life analysis results of ACP52C peptides.
FIG. 7 shows the construction of C16 fatty acid binding peptides for improved in vivo stability.
Fig. 8a to d show the results of cell growth analysis depending on treatment with C 16 fatty acid binding peptide (ACP 52C; ACP52CG; ACP52 CK) in various cancer cell lines.
Fig. 9 a-C show the results of cell growth analysis depending on treatment with C 16 fatty acid binding peptide (ACP 52C; ACP52CG; ACP52 CK) in various p53 mutant or null cancer cell lines.
FIGS. 10a to g show the results of cell growth analysis depending on treatment with ACP52CGK in the normal cell line (BEAS 2B, hMSC) and the cancer cell line (Hep 3B, hs746T, caov-3, MDA-MB-231, U343, HCT116, PANC1, PC3, A549, THP-1).
Fig. 11a to e show the results of anticancer effect analysis of ACP52CG, ACP52CK in Hep3B cell line-xenograft mouse model.
Fig. 12a to d show the results of anticancer effect analysis of ACP52CK in a mouse model of liver cancer induced by DEN treatment.
Fig. 13a to d show the results of analysis of anticancer effects of ACP52CG, ACP52CK in a mice model of liver cancer induced by DEN treatment.
Fig. 14a to d show the results of anticancer effect analysis of ACP52CG, ACP52CK in MDA-MB-231 (LM 1) cell line-xenograft mouse model.
Fig. 15a to j show the results of the analysis of anticancer effect (tumor size and weight, body weight and hematological analysis) and metastasis inhibition effect in Hep3B cell line-xenograft mouse model when treated with ACP52CGK at three different doses at 3 day intervals.
Fig. 16a to k show the results of the analysis of anticancer effect (tumor size and weight, body weight and hematological analysis) and metastasis inhibition effect in Hep3B cell line-xenograft mouse model when treated with ACP52CGK at three different doses at 5 day intervals.
Fig. 17a to j show the results of the analysis of anticancer effect (tumor size and weight, body weight and hematological analysis) and metastasis inhibition effect in MDA-MB-231 (LM 1) cell line-xenograft mouse model when treated with ACP52CGK at three different doses at 3 day intervals.
Fig. 18a to k show the results of the analysis of anticancer effect (tumor size and weight, body weight and hematological analysis) and metastasis inhibition effect in MDA-MB-231 (LM 1) cell line-xenograft mouse model when treated with ACP52CGK at three different doses at 5 day intervals.
Fig. 19a to b show the in vivo half-life analysis results using the fluorescently labeled ACP52 CGK.
Figures 20 a-b show the results of analysis of migration pathways, intracellular localization and intracellular residence time of ACP52CGK in MDA-MB-231 cell lines over time.
Fig. 21 shows the analysis results of investigating whether antibodies against ACP52C, ACP CG, ACP52CK and ACP52CGK were formed by ELISA.
Fig. 22 a-e show burst and repeated in vivo toxicity test results for ACP 52C.
Fig. 23 shows the repeated toxicity test results of ACP52 CGK.
Fig. 24 shows the results of histological analysis of major organs tested for repeat toxicity with ACP52 CGK.
Figures 25a to d show the results of potency analysis of ACP52CGK in cultures of cells derived from tumor tissue of breast cancer patients.
Fig. 26a to d show the results of potency analysis of ACP52CGK in cultures of cells derived from cryopreserved breast cancer patient cancer tissue.
Fig. 27a to d show the results of efficacy comparison analysis of ACP52CGK in cells of specific generation (specific generation) cultured from PDX tumor tissue.
Figures 28 a-b show the results of efficacy and resistance assays of ACP52CGK in cells derived from tumor tissue in patients.
FIG. 29 shows the results of an analysis of the expression of a CP2C transcriptional activity-independent pathway protein in a lung cancer cell line showing resistance to ACP 52C.
FIGS. 30a to C show the results of ACP52C treatment-dependent expression analysis of CP2C transcriptional activity-independent pathway proteins in lung cancer cell lines (A549, PC9, KCL 22).
FIGS. 31a to b show the results of MDM2 overexpression dependent expression analysis of CP2c transcriptional activity independent pathway proteins in lung cancer cell lines (A549, PC 9).
Fig. 32a to d show ACP52C treatment-dependent cell growth analysis results of a549 cell line in which MDM2 overexpression was induced for a long period.
FIGS. 33a to d show MDM2p60 expression models and lung cancer cell line specific alternative splicing and SNP analysis results.
Fig. 34a to d show the results of potency analysis in ACP52C resistant cells depending on the combined treatment of caspase 2 inhibitor and ACP 52C.
Fig. 35 shows changes in cell morphology in ACP52C resistant cells depending on the combined treatment of caspase 2 inhibitor and ACP 52C.
Detailed Description
Hereinafter, the present invention will be described in more detail with reference to the following examples. It should be understood that the examples are included merely for purposes of illustration and are not intended to limit the spirit of the invention in any way.
PREPARATION EXAMPLE 1 preparation of CP2c targeting peptide conjugated to cell penetrating peptide
The transcription factor CP2c is known to be overexpressed in a variety of cancers. According to the study of the American group, inhibition of CP2c expression in liver cancer cell lines was reported to inhibit cell growth, whereas overexpression of CP2c resulted in cancer exacerbation and metastasis [Grant et al.,Antiproliferative small-molecule inhibitors of transcription factor LSF reveal oncogene addiction to LSF in hepatocellular carcinoma,Proc.Natl.Acad.Sci.2012;109(12)：4503-4508].
The present inventors identified peptides [Kang et al.,Identification and characterization of four novel peptide motifs that recognize distinct regions of the transcription factor CP2,FEBS Journal 2005;272：1265-1277], binding to transcription factors CP2C (also referred to as Tfcp, LSF, LBP1, UBP1, etc.) by phage display, selected one type of peptide (CP 2C-targeting peptide, SEQ ID NO: 2) derived from a peptide interfering with DNA binding of CP2C among the identified peptides, and synthesized ACP52C leader substance by binding an internalized RGD (iggd) peptide consisting of 9 amino acids (9 aa)' CRGDKGPDC (Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys, SEQ ID NO: 3) as a Cell Penetrating Peptide (CPP) to the CP 2C-targeting peptide (fig. 7).
EXAMPLE 1 determination of anticancer Effect of ACP52C
The selected peptide binds to CP2c and inhibits the formation of a transcription factor complex comprising CP2c (CP 2c homotetramer and CP2c/CP2b/PIAS1 heterohexamer), thereby indirectly interfering with DNA binding of CP 2c. As a result of analyzing the anticancer effect of the targeted synthesized ACP52C in various cancers, it was confirmed that it showed cancer cell-specific growth inhibition and cell death efficacy (fig. 1).
The growth inhibition and cell death induction efficacy of ACP52C was confirmed by FACS analysis performed by treating cells with ACP52C after synchronizing the cell cycle to the G1/S phase by the double thymidine blocking method. As a result, it was confirmed that polyploidy was formed when the cell cycle was blocked in the G2/M phase. On the other hand, it has been demonstrated that when ACP52C is applied to a cell line synchronized to the G2/M phase with thymidine/nocodazole (nocodazole) treatment, the cell cycle is arrested in the sub-G1 phase and cell death is induced (fig. 2a to d).
It was demonstrated that ACP 52C-induced G2/M phase arrest was caused by increased expression of CHK1/2 protein and decreased expression of Cdc25C, CDK1 and cyclin B proteins, whereas induction of cell death was caused by increased expression of pro-apoptotic proteins and decreased expression of anti-apoptotic marker proteins, as well as apoptosis by caspase activation (fig. 3a to C, fig. 4a to C).
Subcellular movement, localization and stability of ACP52C in cells treated with Cy 5-labeled peptide (Cy 5-ACP 52C) for 30min was analyzed over time by confocal microscopy. Most peptides pass through the cytoplasm, are located in the nucleus from 4 hours, move back to the cytoplasm at 8 hours, and degrade in the cytoplasm at 16 hours. It was confirmed that Cy5-ACP52C peptide was co-localized in the nucleus with CP2C 1 hour after treatment, and CP2C also tended to be distributed in the cytoplasm together with Cy5-ACP52C 8 hours after treatment (FIG. 5).
After incubating ACP52C in a solution containing 10% serum multiple times and then removing serum proteins with Centricon (mw10,000) prior to HPLC analysis, the half-life of ACP52C in the solution containing serum was analyzed by HPLC (model: UHPLC DIONEX Ultimate 3000; flow = 1.000 ml/min). As a result of the experiment, ACP52C did not degrade completely until 24 hours. Meanwhile, after ACP52C was injected into the mice through the tail vein, blood samples were extracted over time, and the extent of ACP52C degradation in the blood samples was analyzed by HPLC. As a result, the EC50 (the time to retain 50% of the intact ACP 52C) was about 2 hours. In addition, when the residual fluorescence intensity in the mice was measured over time by a real-time imaging procedure in mice injected with Cy 5-labeled peptide (Cy 5-ACP 52C) via tail vein, ACP52C was observed in cancer tissues even 5 days after injection, although the total fluorescence intensity in the mice was halved 7.95 hours after treatment (fig. 6a to h).
PREPARATION EXAMPLE 2 Synthesis of CP2c Targeted peptide-fatty acid conjugates
Our data indicate that although ACP52C shows good anticancer activity, ACP52C may be unstable in vivo. As an effort to improve the in vivo stability of ACP52C, 4 types of albumin affinity C 16 fatty acid (palmitoyl acid) conjugated peptides were synthesized (fig. 7). Each peptide was synthesized with modified N-and C-termini, followed by conjugation of C 16 fatty acids to each peptide.
EXAMPLE 2 determination of in vitro anti-cancer Effect of CP2c targeting peptide-fatty acid conjugates
The anticancer effect of the CP2 c-targeting peptide-fatty acid conjugate synthesized in preparation example 2 was analyzed in various cancer cells. As a result, all 3 types of C 16 fatty acid binding peptides (ACP 52CG, ACP52CK and ACP52 CGK) induced cancer cell-specific cell death similar to the control group (ACP 52C), and the calculated GI 50 values at 48 hours after treatment were similar to or more effective than the control GI 50 values (fig. 8a to d, fig. 10a to g). In addition, cell survival curves and GI 50 values were calculated by MTT assay after treatment with ACP52CG and ACP52CK for multiple cancer cell lines with different p53 mutations. As a result, although the GI 50 value of each cell line showed a deviation, it was 10mM or less, which is significantly different from the result of the normal cell line (about 1,000 mM) (fig. 9a to c). In summary, the anticancer effects of ACP52CG, ACP52CK, and ACP52CGK were similar to those of ACP52C, and the fatty acids bound to the complex did not show any negative effect on cancer cells and normal cells.
Example 3 determination of tumor growth inhibition and general physiological toxicity depending on treatment with CP2 c-targeting peptide-fatty acid conjugate in mouse model transplanted with various cancer cell lines
[3-1] Determination of tumor growth inhibition and general physiological toxicity of ACP52CG, ACP52CK and ACP52GK in mouse models transplanted with liver and breast cancer cell lines
To analyze the anticancer efficacy of the CP2 c-targeting peptide-fatty acid conjugate synthesized in preparation example 2 in animal models, ACP52CG and ACP52CK were injected 5 times through the tail vein into Hep3B xenograft mice at 3-day intervals. As a result, both ACP52CG and ACP52CK showed similar efficacy to sorafenib (sorafenib), but less efficacy than ACP 52C. However, no specific abnormalities were found in normal tissues and blood levels, and no toxicity was observed (fig. 11a to e).
In addition, mice that had been subjected to 22 weeks after DEN treatment were divided into 3 groups (7 to 8 mice/group), such as a vehicle-only control group (simulation), sorafenib group (approved drug for liver cancer treatment), and ACP52CG-GFLGE (ACP 52 CK) group. The drug was injected through the tail vein at 3 day intervals at a concentration of 5mg/kg for a total of 12 times. As a result of the analysis, the sorafenib-treated group showed a remarkable anticancer effect (p=0.04) as compared to the control group, and the ACP52CG-GFLGE (ACP 52 CK) -treated group showed an anticancer effect (p=0.001) similar to that of the sorafenib-treated group (fig. 12a to d). Furthermore, the results of potency analysis in mice 15 weeks after DEN treatment for ACP52CG and ACP52CK were also superior to the other control FQI1, but did not show any superior potency compared to ACP52C (fig. 13a to d).
When ACP52CG and ACP52CK were injected 5 times through the tail vein into MDA-MB-231 (LM 1) xenograft mice at 3-day intervals, both ACP52CG and ACP52CK showed tumor regressive efficacy, but the anticancer effect was not significant. No specific abnormalities were found in the blood levels, and therefore no toxicity was observed (fig. 14a to d).
When ACP52GK was injected 5 times through the tail vein into Hep3B xenograft mice at 3-day intervals, followed by analysis of the efficacy, it was confirmed that ACP52GK efficacy was lower than that of ACP52C, but that the efficacy was superior to that of the control FQI1 group.
[3-2] Determination of tumor growth inhibition, metastasis inhibition and general physiological toxicity of ACP52CGK in mouse models transplanted with liver and breast cancer cell lines
ACP52CGK was injected into Hep3B xenograft mice at 3 different concentrations (1.39, 2.77 or 5.54 mg/kg) 5 times through the tail vein at 3 day intervals. Tumor volumes were measured every 3 days after drug injection. Tumors and major tissues were excised from mice sacrificed on day 24 following tumor cell injection. Tumors were weighed and fixed with 4% formaldehyde along with major organs. Hematoxylin/eosin staining was performed after making tissue slides by paraffin sections. A basic CBC analysis was performed on the collected blood samples using a Coulter LH 750 blood analyzer. The obtained data were statistically processed using an Excel program.
As a result, ACP52CGK exhibited a tumor inhibitory effect similar to that of ACP52C, and no abnormality was found in normal tissues and blood levels (fig. 15a to d and 15g to j). In addition, according to fig. 15e to f, the mean and standard deviation of the tumor area metastasized to the lung are shown, confirming that ACP52CGK is superior to ACP52C in tumor metastasis inhibition.
ACP52CGK was injected 5 times at 5 day intervals through the tail vein into Hep3B xenograft mice at 3 different concentrations (1.39, 2.77, 5.54 mg/kg) and evaluated for anticancer effects in the same manner as described above. As a result, ACP52CGK at all three concentrations showed tumor inhibition and metastasis inhibition superior to sorafenib and ACP52C treated groups, and the inhibition was concentration-dependent (fig. 16a to g). In addition, ACP52CGK injected at 5-day intervals showed more potent tumor suppression and metastasis suppression than ACP52CGK injected at 3-day intervals (compare fig. 15a to j with fig. 16a to k). As a result of the comprehensive analysis of all the results, 2.77mg/kg was the optimal concentration. In addition, no specific abnormalities were found in the blood level, and thus no toxicity was observed (fig. 16h to k).
The anti-cancer effect of ACP52CGK was analyzed in MDA-MB-231 (LM 1) xenograft mice in the same manner as in Hep3B xenograft mouse model experiments. Similarly, tumor inhibition and metastasis inhibition in ACP52CGK at all three concentrations (1.39, 2.77, 5.54 mg/kg) was superior to that of sorafenib and ACP52C treated groups (fig. 17a to f and 18a to g). When injections were made at 5 day intervals instead of at 3 day intervals, a better anticancer effect was observed, and 2.77mg/kg was the optimal concentration. In addition, no specific abnormality was found in the blood level, and thus no toxicity was observed (fig. 17g to j and 18h to k).
EXAMPLE 4 evaluation of stability and safety of CP2 c-targeting peptide-fatty acid conjugates
To analyze the in vivo half-life of ACP52CGK, cy 5-labeled ACP52CGK was injected into the tail vein of mice, followed by measurement of the fluorescence intensity remaining in the mice over time by a real-time imaging procedure. As a result, the half-life was about 20.2 hours (fig. 19a to b). These results indicate that conjugation with fatty acids results in significantly improved in vivo stability compared to the lead species ACP52C, which exhibits a half-life of 7.95 hours.
To analyze subcellular movement, localization and stability of ACP52CGK in cultured cells, cy 5-labeled ACP52CGK was treated in cell culture medium for 30 minutes, followed by tracking of peptide subcellular movement over time. In addition, subcellular movement was analyzed by confocal microscopy, whether Cy 5-labeled ACP52CGK also migrated to mitochondria (Hsp 60) and lysosomes (LC 3). As a result, ACP52CGK passes through the cytoplasm from 4 hours and is located mainly in the nucleus and comes out of the cytoplasm after 8 hours, some of which are in the mitochondria but eventually migrate to lysosomes and degrade at 16 hours. These subcellular movement, localization and stability phenomena of ACP52CGK are not different from those in ACP52C (fig. 20 a-b).
Meanwhile, ACP52C is presumed not to exhibit immunogenicity because it consists of 15 amino acids. To verify this, the immunogenicity of ACP52CG and ACP52CK was tested directly in the process of obtaining final new drug candidates. Since the final candidate substance may be a C-16 palmitoyl acid conjugated ACP52C peptide that is chemically indistinguishable from ACP52CG and ACP52CK, it is determined whether an antibody is formed on ACP52CG and ACP52 CK. As a result of ELISA analysis of serum isolated from rabbits injected three times with ACP52CG and ACP52CK, neither rabbit serum induced an immune response on ACP52CG, ACP52CK, and ACP52CGK, including ACP 52C. Therefore, it was concluded that the final candidate substance (ACP 52 CGK) also did not show immunogenicity in humans (fig. 21).
Repeated toxicity tests of ACP52CGK in vivo administration were performed with female and male mice. As a preliminary experiment, when 100mg/kg and 1000mg/kg of ACP52C were repeatedly administered twice, all mice survived, and no hepatotoxicity or adverse reaction to the main organ was observed. As a comprehensive experiment, weight change, water intake, and major histological analysis of organs, organ weight, hematological tests, and blood biochemical tests were analyzed after intravenous infusion of ACP52CGK to solvent and high dose groups (100 mg/Kg) of 6 males and females each at 3-day intervals for 28 days. In summary, no abnormal findings were detected (fig. 22a to e, 23 and 24, and tables 1,2,3 and 4).
[ Table 1]
TABLE 1 absolute organ weights (g) of mice treated with ACP52CGK or untreated with ACP52CGK
[ Table 2]
TABLE 2 treatment with ACP52CGK or without ACP52CGK relative organ weight (%)
[ Table 3]
TABLE 3 hematology of mice treated with ACP52CGK or untreated with ACP52CGK
[ Table 4]
TABLE 4 serum biochemistry of mice treated with ACP52CGK or untreated with ACP52CGK
To investigate whether the efficacy of ACP52CGK tested on cancer cell lines and xenograft mouse models could equally apply to actual clinical practice, the efficacy of ACP52CGK was evaluated in cells cultured from tumor tissue in the fresh state of breast cancer patients. As a preliminary result, ACP52CGK was confirmed to induce cell death at GI50: about 2 μm, similar to those in cancer cell line experiments (fig. 25a to d). Therefore, the cryopreserved cancer tissue of the breast cancer patient is thawed and primary cultured. When the effect of ACP52CGK was analyzed on the cells obtained therefrom, it was also confirmed that cell death was induced at GI50: about 2. Mu.M (FIGS. 26a to d).
EXAMPLE 5 analysis of the cause of resistance of cells exhibiting resistance to CP2 c-targeting peptide-fatty acid conjugate
As a result of analyzing the efficacy of ACP52CGK in each generation of cells originally cultured from PDX (patient-derived xenograft) tumor tissue, no significant difference in sensitivity to ACP52CGK was confirmed in cells derived from the same origin (fig. 27a to d). However, primary cultured cells from some PDX tumor tissues showed resistance to ACP52CGK (fig. 28 a-b).
Cells exhibiting resistance to ACP52CGK (predominantly lung cancer cell lines) tend to exhibit low expression levels of MDM2p90 and relatively high expression of MDM2p 60. Indeed, reduced expression of YY1 in the three lung cancer cell lines treated with ACP52C (a 549, PC9, KCL 22) was confirmed by immunoblotting, but the expression of p53, p63, and p73 did not show any change. The expression of MDM2 (p 90 and p 60) was not reduced in these cell lines. Since MDM2p60 is known to be a protein in which the C-terminal region of MDM2p90 containing the site phosphorylated by ATM (S386, S395 and S407) is deleted, MDM2 degradation is presumed not to be properly regulated by ACP52C treatment (fig. 29, fig. 30a to C).
Accordingly, MDM2p90 was continuously overexpressed by treatment with doxycycline (doxycycline) for 3 weeks, and expression of p53 protein and YY1 was reduced week by week in CP2c transcriptional activity-dependent and independent marker proteins, interestingly by immunoblotting. Meanwhile, since the cell death induced according to ACP52C treatment was analyzed by MTT assay, it was confirmed that the cell death was induced from the second week. Thus, low expression of MDM2p90 in lung cancer cell lines was demonstrated to be responsible for ACP52C resistance (fig. 31 a-b, fig. 32 a-d).
Two models have been proposed for the production of MDM2p60 due to caspase 2 cleavage or alternative splicing of MDM2p 90. When RT-PCR is performed using primer sets capable of detecting alternatively spliced forms of MDM2 in a variety of cancer cell lines, no lung cancer cell-specific spliced forms are found. In addition, when the nucleotide sequence analysis of genomic DNA was tested after PCR cloning to determine whether MDM2p60 was generated from mRNA truncated due to the presence of SNP in the MDM2 gene specifically directed to lung cancer cell lines, no lung cancer cell specific SNP was observed (fig. 33a to d).
Meanwhile, when caspase 2 inhibitor (AC-VDVAD-CHO) was treated to identify a phenomenon occurring due to high activity of caspase 2, particularly in lung cancer cells, it was found that the protein amount of MDM2p90 was increased by immunoblotting, while the protein amount of MDM2p60 was decreased according to the treatment with caspase 2 inhibitor. Thus, we conclude that the over-expressed form of MDM2p60 in lung cancer cells is a result of cleavage of MDM2p90 by caspase 2. Based on these results, cell death induction was analyzed by treating cells (A549 and PDX cells, mammary gland F0-JOS) exhibiting resistance to ACP52C with a combination of caspase 2 inhibitor and ACP 52C. As a result, efficacy was observed at around 1 μm in GI50 in the group treated with the combination of caspase 2 inhibitor and ACP52C (fig. 34a to d, fig. 35).
Thus, the ACP52CGK peptide improved in vivo stability compared to existing ACP52C peptides, and showed growth inhibition/cell death as in ACP52C in all cancer cells, but had no significant effect on normal cells. In addition, it was demonstrated that cancer cells exhibiting resistance to ACP52C can be killed by combined treatment of ACP52C with caspase 2 inhibitors.
<110> Industry-University Cooperation Foundation Hanyang University
<120> CP2 c-targeting anticancer peptides
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<150> KR 10-2019-0085790
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